Assessing the Role of the Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1) Three-finger Domain in Binding Lipoprotein Lipase

Abstract

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) is an endothelial cell protein that transports lipoprotein lipase (LPL) from the subendothelial spaces to the capillary lumen. GPIHBP1 contains two main structural motifs, an amino-terminal acidic domain enriched in aspartates and glutamates and a lymphocyte antigen 6 (Ly6) motif containing 10 cysteines. All of the cysteines in the Ly6 domain are disulfide-bonded, causing the protein to assume a three-fingered structure. The acidic domain of GPIHBP1 is known to be important for LPL binding, but the involvement of the Ly6 domain in LPL binding requires further study. To assess the importance of the Ly6 domain, we created a series of GPIHBP1 mutants in which each residue of the Ly6 domain was changed to alanine. The mutant proteins were expressed in Chinese hamster ovary (CHO) cells, and their expression level on the cell surface and their ability to bind LPL were assessed with an immunofluorescence microscopy assay and a Western blot assay. We identified 12 amino acids within GPIHBP1, aside from the conserved cysteines, that are important for LPL binding; nine of those were clustered in finger 2 of the GPIHBP1 three-fingered motif. The defective GPIHBP1 proteins also lacked the ability to transport LPL from the basolateral to the apical surface of endothelial cells. Our studies demonstrate that the Ly6 domain of GPIHBP1 is important for the ability of GPIHBP1 to bind and transport LPL.

GPIHBP1, an endothelial cell transporter for lipoprotein lipase

“…10 ) ( 26 ). These GPIHBP1 mutants also lacked the capacity to transport LPL from the basolateral to the apical surface of cultured endothelial cells ( 26 ). The fact that the Ly6 domain proved to be important for LPL binding was not entirely unexpected, given that the Ly6 domains of other Ly6 proteins, for example, those of UPAR and CD59, are crucial for ligand binding ( 37,63 ).…”

“…The fact that the Ly6 domain proved to be important for LPL binding was not entirely unexpected, given that the Ly6 domains of other Ly6 proteins, for example, those of UPAR and CD59, are crucial for ligand binding ( 37,63 ). In the case of UPAR, many of the residues involved in ligand binding are located within fi nger 2 of the Ly6 domain ( 37,47,(64)(65)(66), as was the case for GPIHBP1 ( 26 ).…”

“…Thus, intact disulfi de bonds-and the maintenance of the threefi ngered structure of GPIHBP1's Ly6 domain-is critical for LPL binding. In a more recent study, Beigneux et al ( 26 ) changed each amino acid in human GPIHBP1's Ly6 antibody, into the quadriceps of wild-type and Gpihbp1…”

“…We now know that glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1, (GPIHBP1), a GPI-anchored protein of capillary endothelial cells, binds LPL in the subendothelial spaces and transports it to the capillary lumen ( 24 ). The binding of LPL to GPIHBP1 is highly specifi c; even single amino acid substitutions in GPIHBP1 or LPL can abolish their interaction, leading to impaired lipolysis and hypertriglyceridemia (25)(26)(27)(28)(29). We discuss each of these topics in this review.…”

High-resolution imaging of dietary lipids in cells and tissues by NanoSIMS analysis

“…We previously generated a rat hybridoma (11A12) that secretes a monoclonal antibody against mouse GPIHBP1 (16)(17)(18)(19). We grew hybridoma 11A12 in arginine-, leucine-, and lysine-deficient medium (Sigma-Aldrich D9443) supplemented with 15 Nlabeled arginine, leucine, and lysine (Sigma-Aldrich 600113, 340960, 609021).…”